C1r, subunit of the first complement component: purification, properties, and assay based on its linking role
The Journal of Clinical Investigation
|April 1, 1971
Summary
A novel purification method yields highly pure C1r, a complement system component. This advance enables the development of specific antibodies and further research into C1r's function and stability.
Area of Science:
- Immunology
- Biochemistry
Background:
- The first complement component (C1) is crucial for immune responses.
- C1r, a subunit of C1, plays a key role in complement activation.
- Obtaining highly purified C1r has been a challenge for researchers.
Purpose of the Study:
- To describe a novel method for purifying C1r.
- To characterize the purified C1r.
- To develop a sensitive assay for C1r activity.
Main Methods:
- Stepwise purification involving euglobulin precipitation, ion-exchange chromatography (DEAE- and CM-cellulose), and preparative polyacrylamide electrophoresis.
- Analytical polyacrylamide electrophoresis and immunodiffusion for purity assessment.
- Hemolytic assay for C1r activity, measuring its ability to link C1s to C1q.
Main Results:
- A highly purified C1r preparation was obtained, free of C1q and C1s activities, showing a single band on electrophoresis.
- Antisera against purified C1r showed a single precipitation band, indicating monospecificity.
- C1r activity was found to be labile at 37°C, pH 7-8, and low ionic strength.
- Electrophoretic mobility indicated C1r is a beta-globulin; molecular weight was estimated at 168,100 via Sephadex chromatography.
- A sensitive hemolytic assay was developed, demonstrating a stoichiometric relationship between C1r concentration and C1 complex formation.
Conclusions:
- A robust method for C1r purification and characterization has been established.
- The developed assay allows for sensitive detection and quantification of C1r.
- These findings facilitate further studies on C1r's role in the complement system and the development of specific reagents.
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